In Vitro Antibacterial Activity of Several Plant Extracts and Oils against Some Gram-Negative Bacteria.

Background: Medicinal plants are considered new resources for producing agents that could act as alternatives to antibiotics in the treatment of antibiotic-resistant bacteria. The aim of this study was to evaluate the antibacterial activity of 28 plant extracts and oils against four Gram-negative bacterial species. Methods: Experimental, in vitro, evaluation of the activities of 28 plant extracts and oils as well as some antibiotics against E. coli O157:H7, Yersinia enterocolitica O9, Proteus spp., and Klebsiella pneumoniae was performed. The activity against 15 isolates of each bacterium was determined by disc diffusion method at a concentration of 5%. Microdilution susceptibility assay was used in order to determine the minimal inhibitory concentrations (MICs) of the plant extracts, oils, and antibiotics. Results: Among the evaluated herbs, only Origanum syriacum L., Thymus syriacus Boiss., Syzygium aromaticum L., Juniperus foetidissima Wild, Allium sativum L., Myristica fragrans Houtt, and Cinnamomum zeylanicum L. essential oils and Laurus nobilis L. plant extract showed anti-bacterial activity. The MIC50 values of these products against the Gram-negative organisms varied from 1.5 (Proteus spp. and K. pneumoniae( and 6.25 µl/ml (Yersinia enterocolitica O9 ) to 12.5 µl/ml (E. coli O:157). Conclusion: Among the studied essential oils, O. syriacum L., T. syriacus Boiss., C. zeylanicum L., and S. aromaticum L. essential oils were the most effective. Moreover, Cephalosporin and Ciprofloxacin were the most effective antibiotics against almost all the studied bacteria. Therefore, O. syriacum L., T. syriacus Boiss., C. zeylanicum L., and S. aromaticum L. could act as bactericidal agents against Gram-negative bacteria.

preservation, 2 pharmacy, and medicine. 3,4 They are expected to form new sources of antimicrobial drugs, especially against bacteria. 5 The antibacterial effectiveness of aromatic oils has been divided into a good, medium, or bad. 6,7 These oils can also produce some defense products against several natural enemies. 8 In addition, and in order to continue their natural growth and development, aromatic oils may produce some secondary metabolites in response to some external stress. 9 The extracts and oils of 28 plants used in this work have been traditionally employed by people for various purposes in different parts of the world. Cinnamomum zeylanicum essential oil has antibacterial and antifungal activities 10 as well as anti-diabetic properties; 11 Citrus limon and Rosmarinus officinalis L. essential oils possess antioxidant properties; 12,13 Citrus aurantium has immunological effects in humans; 14 Eucalyptus globulus oil has good antimicrobial activities; 15,16 Thymus pannonicus essential oil has an excellent effect against E. coli O157:H7; 17 light thyme essential oil inhibits the growth of E. coli O157:H7 in foods; 18 Brillantaisia lamium extract exhibits antibacterial and antifungal effects against Staphylococcus aureus, Enterococcus faecalis, Candida tropicalis, and Cryptococcus neoformans; 19 and finally Crinum purpurascens herb extract has antimicrobial activities against Salmonella paratyphi A and B. 20 Traditionally, many plant extracts and oils are used as medicinal plants in Syria for many purposes, particularly for respiratory and gastrointestinal disorders.
The aim of this study was to screen the in vitro antibacterial activity of 28 plant extracts and oils against some Gram-negative bacteria, including: E. coli O157:H7, Yersinia enterocolitica O9, Proteus spp., and Klebsiella pneumoniae.

Essential Oil Extraction
Essential oils from fresh, clean, weighed aerial parts, flowers, leaf fruits, barks, seeds, rhizomes, and bulbs (table 1) extracted by hydro-steam distillation using the Clevenger apparatus were collected and stored in sterile vials. 22 Briefly, 100 to 150 g of each plant was introduced in the distillation flask (1 L), which was connected to a steam generator via a glass tube and to a condenser to retrieve the oil. This was recovered in a funnel tube. Aromatic molecules of the essential oils were released from the plant material and evaporated into hot steam. The hot steam forced the plant material to release the essential oil without burning the plant material itself. Then, steam containing the essential oil was passed through a cooling system in order to condense the steam. The steam was applied for 3 h. After settling the recovered mixture, essential oil was withdrawn. The supernatant essential oil was filtered through anhydrous Na 2 SO 4 to dry the yielded essential oil. Afterward, the essential oil was collected in tightened vials and stored in a refrigerator. For the antimicrobial activity test, several dilutions of the oils were done using dimethyl sulfoxide (DMSO).

Preparation of Ethanolic Extracts
Successive solvent extraction was performed for some plants (table 1). Leaves and bulbs were washed, air dried for 7-8 days, and ground into powder before they were placed into the flask of the Soxhlet apparatus for extraction using ethanol with increasing order of polarity to extract the phytoconstituents separately at 20ºC for 3-4 h. (The ethanol used was HPLC grade obtained from Sigma-Aldrich, Germany.) Whatman No.1 filter papers were then applied to filter the extracts. After that, reduced pressure was applied to evaporate and dry the filtrates, which were stored at -20ºC in labeled, sterile, screw-capped bottles.

Antibacterial Susceptibility Assay
Muller-Hinton Broth (MHB, Merck) medium was used to grow the test isolates for 22 h at 37°C. Final bacterial numbers were standardized to 1×10 6 CFU/ml. A total of 0.1 ml of bacterial suspension was poured on each plate, containing Muller-Hinton Agar (MHA, Merck). The lawn culture was prepared by sterile cotton swab and allowed to remain in contact for 1 min. Thereafter, a 5% concentration of each plant extracts was prepared. The sterile filter paper discs (6-mm diameter) were placed on the lawn cultures, and 24 h after incubation at 37°C, the inhibition zone was measured in mm.

Antibiotics Minimum Inhibitory Concentration Determination
In order to estimate the antibiotics susceptibility, the well broth microdilution method was used with 96-well plates (TPP, Switzerland). The antibiotics were diluted twofold in LB broth ® (Acumedia, Michigan, USA), and the wells were inoculated with 1×10 6 CFU of bacteria (in a 0.2 ml final volume). The incubation period was 24 h at 37°C. The lowest concentration that inhibited 50% of visual growth was recorded and interpreted as the MIC 50 . The MIC testing was performed according to the recommendations of  23 The range of the concentrations assayed for each antibiotic was 0.064 to 128 μg/ml. The absorbance was determined at 590 nm (Thermo-Lab Systems Reader, Finland). All the tests were performed in triplicate and then averaged. The investigated antibiotics were Ciprofloxacin, Levofloxacin, Ofloxacin, Sparfloxacin, Ceftazidime, Ceftriaxone, and Cefotaxime. Positive control was done without adding any antibiotics.

Plants Extracts and Oils Minimum Inhibitory Concentration Determination
The microdilution broth susceptibility assay was used. 24 Three replicates of the serial dilutions of each essential oil were prepared in LB broth medium in 96-well microtiter plates, using a range of concentrations for each essential oil from 0.75 to 50 µl/ml. Next, 100 μl of freshly grown bacteria, standardized until a bacterial number of 1×10 6 CFU/ml in LB broth was achieved, was added to each well. Positive and negative controls were also done. The plate was incubated with shaking for 24 h at 37˚C. The lowest concentration that inhibited 50% of visual growth was recorded and interpreted as the MIC 50 .

Statistical Analysis
Optimal concentrations for the most effective essential oils and plant extracts were estimated by Probit Analysis (SPSS Inc. 2010; Finney, 1971). Minimum concentrations to achieve 50% inhibition of the various bacteria (MIC 50 ) were considered significantly different if their 95% confidential limits did not overlap. Table 2 demonstrates that O. syriacum. L., T. syriacus, S. aromaticum, C. zeylanicum, L. nobilis L., J. foetidissima, A. sativum L., and M. fragrans Houtt. had good antibacterial activities against the Gram-negative bacteria, whereas the rest of the studied extracts were ineffective.

Discussion
Because of their safety and low cost as well as their impact on a large number of microbes, 25 medicinal plants may have the ability to treat bacterial resistance to many types of antibiotics. The antimicrobial effects of aromatic oils extracted from a large number of plants have been evaluated and reviewed, 26,27 and the mechanisms that enable the natural ingredients of herbs and spices to resist microbes have been discussed. 28 The results show that these mechanisms vary greatly depending on the components of the essential oil. 29,30 In the present study, the efficacy of some plant extracts and oils was determined, quantitatively, by measuring the diameter of the inhibition zones around the discs (table 2) 31 it can be logically assumed that the above-mentioned plant extracts and oils have a bactericidal effect on Gram-negative bacteria, especially against Proteus spp. and K. pneumoniae.
Ooi et al. 32 reported that Cinnamomum verum shows excellent activities against E. coli and Proteus vulgaris. Preuss et al. 33 found that origanum essential oil proves cidal to E. coli and K. pneumoniae. In addition, Barbosa et al. 34 found that the MIC 90 of Origanum vulgare essential oil is 0.46% (v/v) against E. coli. López et al. 35 found that 8-10% (v/v) concentrations of Origanum vulgare essential oil can completely inhibit the growth of E. coli and other Gram-negative bacteria. Elsewhere, Mkaddem et al. 36 reported that Mentha essential oils are very active against K. pneumoniae bacteria, whereas they are less effective against E. coli. Furthermore, Mentha longifolia oil is thought to exhibit an antimicrobial activity against some Gram-positive bacteria such as Streptococcus mutans and Staphylococcus aureus, but without affecting Pseudomonas aeruginosa. 37 Since the antibacterial effectiveness of medicinal plants varies dramatically depending on the phytochemical characteristics of plant families and subfamilies, it is not surprising to note the difference in this efficacy even when using samples taken from the same plant, but from two different regions. 38 Our results reveal that the cephalosporins were the most effective antibiotics against almost all the studied bacteria, and only Ciprofloxacin, one of the fluoroquinolones group, was effective against these bacteria.

Conclusion
O. syriacum. L., T. syriacus Boiss., S. aromaticum L., C. zeylanicum L., J. foetidissima Wild, A. sativum L., and M. fragrans Houtt. oils and L. nobilis L. extract were the most effective plant extracts against the Gram-negative bacteria studied in this work. These plant extracts could be a potential source of new antibacterial agents.
Further and more specific studies, in vivo, are recommended to determine the efficacy of these essential oils in the treatment of gram-negative bacterial infections.